Primary ciliary dyskinesia (PCD) is a disease caused by impaired function of motile cilia. PCD mainly affects the lungs and reproductive organs. Inheritance is autosomal recessive and X-linked. PCD patients have diverse clinical manifestations, thus making the establishment of proper diagnosis challenging. The utility of next-generation sequencing (NGS) technology for diagnostic purposes allows for better understanding of the PCD genetic background. However, identification of specific disease-causing variants is difficult. The main aim of this study was to create a unique guideline that will enable the standardization of the assessment of novel genetic variants within PCD-associated genes. The designed pipeline consists of three main steps: (1) sequencing, detection, and identification of genes/variants; (2) classification of variants according to their effect; and (3) variant characterization using in silico structural and functional analysis. The pipeline was validated through the analysis of the variants detected in a well-known PCD disease-causing gene (DNAI1) and the novel candidate gene (SPAG16). The application of this pipeline resulted in identification of potential disease-causing variants, as well as validation of the variants pathogenicity, through their analysis on transcriptional, translational, and posttranslational levels. The application of this pipeline leads to the confirmation of PCD diagnosis and enables a shift from candidate to PCD disease-causing gene.

Introduction: Chronic endoplasmic reticulum (ER) stress and increased apoptosis are involved in the pathogenesis of glycogen storage disease Ib (GSD Ib), whereas small molecule phenylbutyrate (4‐PBA) showed the capability of reducing ER stress‐induced apoptosis. The objective was to generate an in vitro system in which capability of small molecules (SMs) to influence ER stress and apoptosis could be screened at the expression level.Methods: G6PT‐deficient FlpInHEK293 cell line was created and validated using the CRISPR/Cas9 knockout method. Molecular markers of unfolded protein response (ATF4, DDIT3, HSPA5, XBP1s), and apoptosis (BCL2/BAX, CASP3, CASP7) in G6PT‐deficient cells were analyzed using RT‐qPCR method before and upon the treatment with 4‐PBA.Results: Treatment with the most effective dose of 1 mM 4‐PBA reduced the expression of UPR markers and executioner caspases, while increased BCL2/BAX ratio in G6PT‐deficient cells. Our results proved the concept that 4‐PBA could alleviate markers of ER stress detected in the GSD Ib in vitro model system and prevent cell death.Conclusion: This cost‐effective in vitro model screens the therapeutic potential of SMs affecting ER stress and apoptosis in G6PT‐deficient kidney cells, offering a first‐line screening assay for promising compounds. 4‐PBA's potential repurposing for GSD Ib patients opens new research directions.